Pseudophosphorylation of Arabidopsis jasmonate biosynthesis enzyme lipoxygenase 2 via mutation of Ser600 inhibits enzyme activity

Abstract

Jasmonates are oxylipin phytohormones critical for plant resistance against necrotrophic pathogens and chewing herbivores. An early step in their biosynthesis is catalyzed by non-heme iron lipoxygenases (LOX; EC 1.13.11.12). In Arabidopsis thaliana, phosphorylation of Ser⁶⁰⁰ of AtLOX2 was previously reported, but whether phosphorylation regulates AtLOX2 activity is unclear. Here, we characterize the kinetic properties of recombinant WT AtLOX2 (AtLOX2^WT). AtLOX2^WT displays positive cooperativity with α-linolenic acid (α-LeA, jasmonate precursor), linoleic acid (LA), and arachidonic acid (AA) as substrates. Enzyme velocity with endogenous substrates α-LeA and LA increased with pH. For α-LeA, this increase was accompanied by a decrease in substrate affinity at alkaline pH; thus, the catalytic efficiency for α-LeA was not affected over the pH range tested. Analysis of Ser⁶⁰⁰ phosphovariants demonstrated that pseudophosphorylation inhibits enzyme activity. AtLOX2 activity was not detected in phosphomimics Atlox2^S600D and Atlox2^S600M when α-LeA or AA were used as substrates. In contrast, phosphonull mutant Atlox2^S600A exhibited strong activity with all three substrates, α-LeA, LA, and AA. Structural comparison between the AtLOX2 AlphaFold model and a complex between 8R-LOX and a 20C polyunsaturated fatty acid suggests a close proximity between AtLOX2 Ser⁶⁰⁰ and the carboxylic acid head group of the polyunsaturated fatty acid. This analysis indicates that Ser⁶⁰⁰ is located at a critical position within the AtLOX2 structure and highlights how Ser⁶⁰⁰ phosphorylation could affect AtLOX2 catalytic activity. Overall, we propose that AtLOX2 Ser⁶⁰⁰ phosphorylation represents a key mechanism for the regulation of AtLOX2 activity and, thus, the jasmonate biosynthesis pathway and plant resistance.

Keywords: 13S-lipoxygenase 2; enzyme kinetics; enzyme structure; jasmonate; lipoxygenase pathway; phosphorylation; protein structure prediction; site-directed mutagenesis.